Tartrate salt of isofagomine and methods of use

ABSTRACT

A novel tartaric acid salt of Isofagomine (Isofagomine tartrate) that can be used for the treatment of Gaucher disease is provided. The invention also provides a crystalline form of isofagomine tartrate, method for preparing the salt, a pharmaceutical composition containing the salt, and a method of treating Gaucher disease.

CROSS-REFERENCED RELATED APPLICATIONS

This application is a divisional application of 11/752,658, filed May23, 2007 and claims priority from U.S. Provisional Patent ApplicationNos. 60/808,020, filed May 24, 2006, and 60/890,719, filed Feb. 20,2007, all of which are incorporated by reference herein in theirentirety.

BACKGROUND OF THE INVENTION

Gaucher disease is a lysosomal storage disorder that is associated withthe accumulation of glycosphingolipids (GSL) in cells, particularlymonocytes and macrophages, of afflicted individuals. This aberrant buildup of GSL results from a genetic deficiency (mutation) in the geneencoding the lysosomal enzyme acid β-glucosidase (glucocerebrosidase;GCase), the lysosomal hydrolase that breaks down the GSLglucosylceramide (GluCer). The majority of glucocerebrosidase gene (Gba)mutations cause the GCase protein to misfold in the endoplasmicreticulum (ER). Misfolded GCase is recognized by the ER quality controlsystem and subsequently degraded instead of being processed andtrafficking to the lysosome.

Gaucher disease is pan-ethnic, with an overall disease frequency ofabout 1 in 50,000-100,000 births. Certain populations have a higherprevalence. In the Ashkenazi population, for example, about 1 in 15people are carriers for a Gba mutation. According to the NationalGaucher Foundation, about 2,500 Americans suffer from Gaucher disease.

Gaucher disease is an autosomal recessive disorder and is the mostcommon lysosomal storage disease. The disease has been classified intothree clinical types, depending on neurological involvement and diseaseseverity. Type 1 is the most common and is characterized by an absenceof neurological involvement. Patients exhibit a broad spectrum ofseverity; some can remain asymptomatic throughout life. Most Type 1patients exhibit enlargement of the spleen and liver, skeletalabnormalities and bone lesions, and sustained inflammatory reactions.Hepatic glucocerebroside levels are elevated from 23-fold to 389-foldabove normal levels in Type 1 Gaucher patients.

Type 2 Gaucher disease is the rarest and most severe form. It isassociated with early onset of acute neurologic disease. Thecharacteristic feature of neuronopathic Gaucher disease is anabnormality of horizontal gaze. Afflicted patients develop progressiveencephalopathy and extrapyrimidal symptoms such as rigidity andParkinson's-like movement (parkinsonism). Most Type 2 Gaucher patientsdie in early childhood from apnea or aspiration due to neurologicaldeterioration.

Type 3 Gaucher disease also has neurological involvement, although to alesser extent than Type 2. These patients also have thehepatosplenomegaly and skeletal defects characteristic of Type 1, aswell as central nervous system symptoms that include poor coordinationof movements (ataxia), seizures, paralysis of the eye muscles, epilepsy,and dementia. Patients with Type 3 Gaucher disease can live intoadulthood, but may have a shortened life span. Three sub-classificationsof Type 3 have been reported: Type 3a, which is associated withprominent hepatosplenomegaly and bone marrow disease; Type 3b, which isassociated with limited systemic symptoms; and Type 3c, which isassociated with hepatosplenomegaly, corneal opacities, progressiveataxia and dementia, and cardiac valve and aortic root calcification.

Approaches for the treatment of Gaucher disease include enzymereplacement therapy (ERT), bone marrow transplants (BMT), substratereduction therapy (SRT), gene therapy, and pharmacological chaperonetreatment. Isofagomine is a potent inhibitor of recombinant human acidβ-glucosidase (GCase). Pharmacological chaperone methods for enhancingmutant enzyme activities in lysosomal storage disorders using enzymeinhibitors such as isofagomine are disclosed in commonly owned U.S. Pat.Nos. 6,916,829; 6,599,919; 6,589,964; 6,583,158, and 7,141,582 each ofwhich are herein incorporated by reference in their entirety. Forexample, the addition of an inhibitor of GCase to a fibroblast culturemedium has been shown to lead to an increase in the trafficking andlysosomal activity of GCase, indicating that such an inhibitor may be oftherapeutic interest in the treatment of Gaucher disease.

It has recently been discovered that there is a link between mutationsin the Gba gene and Parkinson's disease. In one study, a group of 17patients with rare, early onset, treatment-resistant parkinsonism werefound to have at least one allele with a Gba missense mutation,including homozygous and heterozygous individuals for N370S, a mutationtypically associated with type 1, non-neuronopathic disease (Tayebi etal., Mol. Genet. Metab. 2003; 79; 104-109). In another study, apopulation of 99 Ashkenazi Jews with idiopathic Parkinson's disease wereevaluated for six Gba mutations (N370S, L444P, 84GG, V394L, and R496H).Thirty-one Parkinson's patients had one or two mutant Gba alleles: 23were heterozygous for N370S; 3 were homozygous for N370S; 4 wereheterozygous for 84GG; and 1 was heterozygous for R496H (Aharon-Peretzet al., New Eng. J. Med. 2004; 351: 1972-77). The frequency of a mutantN370S allele was 5 times that among 1573 normal subjects, and that of84GG was 21 times that of normal subjects. Among patients withParkinson's disease, patients carrying a Gba mutation also were youngerthan those who were not carriers. This study suggests thatheterozygosity for a Gba mutation may predispose Ashkenazi Jews toParkinson's disease. Since isofagomine has been shown to cross theblood-brain barrier in animals, and increases the activity of bothmutant wild-type GCase, it can be used to treat both Parkinson'spatients who have a heterozygous mutation in GCase, or who are at riskfor developing Parkinson's disease due to other factors, but who maybenefit from increased levels of wild-type GCase.

Although the compound of isofagomine is a potent and selectiverecombinant human acid β-glucosidase (GCase) inhibitor, its use inpharmaceutical products presents challenges. For example, thehydrochloride salt of isofagomine (isofagomine-HCl) is disclosed in U.S.Pat. No. 5,844,102. However, isofagomine-HCl as well as isofagomine freebase are not readily purified on a large scale and have poor solid stateproperties for use in an industrial scale manufacturing processes andpharmaceutical formulations.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a compound of atartaric acid salt of isofagomine or isofagomine tartrate, representedby the following chemical structure:

wherein n is 1 or 2. The invention also provides isofagomine tartratewith a high purity and in a crystalline form.

In another embodiment, the invention provides a composition containingisofagomine tartrate, preferably at least 50%, preferably, 90%, and evenmore preferably, 99%. The invention also provides a compositioncontaining at least 90% or more of isofagomine tartrate where 90% of theisofagomine tartrate has a particle size of 1200 μm.

In other embodiment, the invention provides a pharmaceutical compositioncontaining isofagomine tartrate and one or more pharmaceuticallyacceptable excipients.

In other embodiment, the invention provides a method for the preparationof an isofagomine tartrate. A method for preparing a highly purifiedisofagomine tartrate is also provided.

Yet, in another embodiment, the invention provides a method of treatingGaucher disease in a mammal by enhancing GCase activity in the mammal byadministrating pharmaceutically effective amount of isofagomine tartrateor its pharmaceutical compositions.

In another embodiment, the invention provides L-(+)-tartaric acid saltof isofagomine. The invention also provides a complex of a tartaric acidand isofagomine.

In another embodiment, the invention provides a crystalline form ofisofagomine tartrate. Preferably, the crystalline form has an x-raypowder diffraction pattern that includes five or more peaks of thefollowing peaks: (2 theta) 9.29±0.009, 14.17±0.009, 16.34±0.009,18.07±0.009, 18.72±0.009, 19.44±0.009, 20.56±0.009, 22.13±0.009,23.01±0.009, 24.54±0.009, and 27.12±0.009. More preferably, the x-raypattern includes the following peaks: (2 theta) 9.29, 14.17, 16.34,18.07, 18.72, 19.44, 20.56, 22.13, 23.01, 24.54, and 27.12. Even morepreferably, the crystalline form has an x-ray powder diffraction patternthat is substantially the same as the pattern shown in FIG. 5.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a mass spectrum using positive ESI for isofagomine tartrateprepared according to one embodiment of the present invention.

FIG. 2 shows an ¹H NMR in D₂O of isofagomine tartrate prepared accordingto one embodiment of the present invention.

FIG. 3 show a ¹³C NMR in D₂O of isofagomine tartrate prepared accordingto one embodiment of the present invention.

FIG. 4 shows a spin-echo ¹³C NMR in D₂O of isofagomine tartrate preparedaccording to one embodiment of the present invention.

FIG. 5 shows an X-ray powder diffraction pattern of isofagomine tartrateprepared according to one embodiment of the present invention.

FIG. 6 shows a thermo-gravimetric analysis (TGA) of isofagomine tartrateprepared according to one embodiment of the present invention.

FIG. 7 shows an infrared spectrum of isofagomine tartrate preparedaccording to one embodiment of the present invention.

FIG. 8A shows an ¹H NMR (D₂O) of Isofagomine L-(+)-Tartrate salt (2:1)prepared according to one embodiment of the present invention.

FIG. 8B shows an ¹H NMR (D₂O) of Isofagomine D-(−)-Tartrate salt (2:1)prepared according to one embodiment of the present invention.

FIG. 8C shows an ¹H NMR (D₂O) of Isofagomine D-(−)-Tartrate salt (1:1)prepared according to one embodiment of the present invention.

FIG. 9 shows Results of IFG tartrate on GCase activity of healthyindividuals.

FIG. 10 shows a particle size distribution analysis result ofisofagomine prepared according to one embodiment of the presentinvention.

DETAILED DESCRIPTION

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention as well as how to make and use them.

The term “Gaucher disease” includes Type 1, Type 2 and Type 3 (including3a, 3b and 3c), and intermediates and subgroups thereof based onphenotypic manifestations.

The terms “effective amount” and “amount effective” refer to the amountthat is sufficient to result in a therapeutic response. A therapeuticresponse may be any response that a user (e.g., a clinician) willrecognize as an effective response to the therapy, includingimprovements in the foregoing symptoms and surrogate clinical markers.Thus, a therapeutic response will generally be an amelioration of one ormore symptoms of Gaucher disease, e.g., amelioration of progressiveneurodegeneration in Types 2 and 3 Gaucher patients. The“therapeutically effective amount” will vary depending on theformulation used, the type of Gaucher disease and its severity, and theage, weight, physical condition and responsiveness of the mammal to betreated. A therapeutic response will also be an amelioration of one ormore, symptoms of Parkinson's disease, or other α-synucleinopathies suchas Lewy Body Dementia, for which isofagomine tartrate is contemplatedfor treatment.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce untoward reactions at an unacceptable level when administered toa human. Preferably, as used herein, the term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans.

The term “carrier” applied to pharmaceutical compositions of theinvention refers to a diluent, excipient, or vehicle with which theIsofagomine tartrate is administered. The choice of carrier can beselected with regard to the intended route of administration andstandard pharmaceutical practice. The pharmaceutical compositions maycomprise as—or in addition to—the carrier any suitable binder(s),lubricant(s), suspending agent(s), coating agent(s), solubilizingagent(s). Such pharmaceutical carriers can be sterile liquids, such aswater, saline solutions, aqueous dextrose solutions, aqueous glycerolsolutions, and oils, including those of petroleum, animal, vegetable, orsynthetic origin, such as peanut oil, soybean oil, mineral oil, andsesame oil. Suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin, 18th Edition. Inone particularly preferred embodiment of the present invention, thecarrier is suitable for immediate-release, e.g., release of most or allof the active ingredient over a short period of time, such as 60 minutesor less, and make rapid absorption of the drug possible.

A “pharmaceutically acceptable carrier” means a carrier that is usefulin preparing a pharmaceutical composition that is generally safe,non-toxic, and neither biologically nor otherwise undesirable, andincludes an excipient that is acceptable for pharmaceutical use. A“pharmaceutically acceptable carrier” as used in the present applicationincludes both one and more than one such carrier.

The term “hydroxyl protecting group” refers to any common protectinggroup for hydroxyl to avoid undesired reactions, such as, but notlimited to, methoxymethyl, 4-methoxybenzyl, benzyl,dimethylisopropylsilyl, trimethylsilyl, and alkyl carbonyl.

“Individuals” refers to mammals, preferably humans, domestic animals,rodents or primates, and most preferably humans.

An “individual in need of treatment” is an individual that hasdeveloped, or is likely to develop, Gaucher disease or anα-synuclienopathy such as Parkinson's disease. In one embodiment, theindividual is a member of the Ashkenazi Jewish population who has beendiagnosed with or who has been identified as having an increased risk ofdeveloping Gaucher disease due to inherited mutations in the Gba gene.However, the term “individual” encompasses anyone in the world having,or genetically at risk of developing, Gaucher disease, or having at riskof developing an α-synucleinopathy such as Parkinson's disease.

As used herein, the term “enhancing” the activity GCase meansstabilizing a conformation of a mutant GCase protein in the ER so thatit i) folds in a conformation which permits it to exit the ER, resultingin increased levels of GCase in the ER, and/or ii) achieves its nativelocation in the cell, and/or iii) exhibits catabolic activity towardscerebroside, its lipid substrate. This term also refers to increasing orprolonging the activity of an exogenously administered GCase protein,i.e., by increasing the stability and extending the in vivo half-life ofGCase, thus, prolonging its activity.

The phrase “substantially pure,” as used herein means that theisofagomine salt contains no more than about 5% of another compound.Preferably, the “substantially pure” isofagomine salt contains about 2%or less of any other compound. Even more preferably, the “substantiallypure isofagomine salt contains about 1% or less of any other compound.

The terms “about” and “approximately” shall generally mean an acceptabledegree of error for the quantity measured given the nature or precisionof the measurements. Typical, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values. Alternatively, and particularly inbiological systems, the terms “about” and “approximately” may meanvalues that are within an order of magnitude, preferably within 10- or5-fold, and more preferably within 2-fold of a given value. Numericalquantities given herein are approximate unless stated otherwise, meaningthat the term “about” or “approximately” can be inferred when notexpressly stated.

As used herein, the singular forms “a,” “an,” and “the,” include theplural unless the context clearly indicates otherwise. Thus, forexample, reference “a” carrier includes one or more carriers.

In accordance with the present invention, a specific form ofisofagomine, Isofagomine tartrate, is provided. Isofagomine tartrate hasmany improved characteristics compared with previously described formsof isofagomine. For example, isofagomine tartrate is more easilypurifiable, especially in solvents such as water and/or ethanol, and hasgreater stability than other known salt forms of isofagomine.Isofagomine tartrate is particularly suitable for industrial scaleproduction, e.g., production of greater than 1 Kg of product.

Isofagomine is (3R,4R,5R)-5-(hydroxymethyl)-3,4-piperidinediol havingthe following chemical structure:

It has a molecular formula of C₆H₁₃NO₃ and a molecular weight of 147.17g/mol. Synthesis of this compound is described in U.S. Pat. Nos.5,844,102 to Sierks et al. and 5,863,903 to Lundgren et al. The '102patent discloses that the compounds described therein can be combinedwith pharmaceutically acceptable salts, including salts of organiccarboxylic acid salts such as acetic, lactic, tartaric, malic,isothionic, lactobionic, and succinic acids. However, the only saltexemplified in this patent (and in subsequent literature of which theapplicant is aware) is the hydrochloride salt. As described herein, thehydrochloride salt is not suitable for industrial production or forformulation in dosage forms.

The term isofagomine tartrate used herein means a tartaric acid salt ofisofagomine and can be represented as follows:

wherein n is 1 or 2. Tartaric acid could have different stereoisomericforms; D- or L-tartaric acid, or DL- or meso-tartaric acid. The presentinvention, as well as Examples, is mainly described with reference toL-(+)-tartaric acid as a preferred embodiment and the isofagomine saltthereof. However, the term, tartaric acid is intended to cover both Dand L isomers as well as a DL mixture and meso-tartaric acid, and thusthe term isofagomine tartrate is intended to include mono- ordi-isofagomine-L-tartrate, mono- or di-isofagomine-D-tartrate, mono- ordi-isofagomine-DL-tartrate and/or mono- or di-isofagomine-meso-tartrate.L-tartaric acid is (2R,3R)-(+)-tartaric acid with enantiomer enrichmentof 97% or higher, and D-tartaric acid is (2S,3S)-(−)-tartaric acid withenantiomer enrichment of 97% or higher. DL-tartaric acid is a mixture ofD- and L-tartaric acid with enantiomer enrichment of less than 97%.

Isofagomine tartrate may be prepared from isofagomine free basedissolved in alcohol, preferably ethanol, and treated with thesubstituted carboxylic acids, including amino acids, di-carboxylicacids, or tartaric acid, including diacyl tartaric acids, in alcohol,preferably ethanol, with stirring at room temperature. The acid saltprecipitates from the ethanol solution. The crude isofagomine tartrateacid salt is collected by filtration. The isofagomine solution may beprefiltered to remove any particle impurities before the acid is added.After the addition of the acid, the resulting suspension may be cooledfor complete precipitation of the salt.

In another embodiment, the isofagomine acid salt of the presentinvention may be prepared by adding a substituted carboxylic acid ortartaric acid to a solution wherein isofagomine is prepared in situwithout isolation of the isofagomine.

Alternatively, the isofagomine acid salt of the present invention may beprepared from a mineral acid salt of isofagomine such as isofagominehydrochloride. The conversion can be accomplished by generatingisofagomine and subsequently treating the free base with a substitutecarboxylic acid. For example, isofagomine free base can be formed bytreating with the hydrochloride salt a basic source such as a mineralbase, ammonia gas or aqueous ammonium hydroxide solutions, or byexposing it to a solid supported basic resin or a column of basic resin.When a basic resin is used, the resin column can be eluted with water,aqueous ammonium hydroxide or ammonium hydroxide in an alcohol such asmethanol, ethanol, IPA, and the like to provide the isofagimine freebase, which can be converted to the isofagomine acid salt of the presentinvention.

Because tartaric acid is a diacid, conversion to the tartaric acid saltcan be done with a range of acid to base ratios: 0.5 molar equivalentsup to 1 molar equivalent of tartaric relative to isofagomine free base.Tartaric acid can be racemic (the D or -L form) or one of threestereoisomeric forms, the L-(+) form, the D-(−) form, and the meso form.Preferred conditions for making the tartrate salt use ammonium hydroxidesolution to generate the free base, 9:1 ethanol/ammonium hydroxide toelute the free base on a silica gel column, evaporation of solvent andexcess ammonium hydroxide, formation of the tartrate salt inwater/ethanol, and crystallization from water/ethanol.

Isofagomine and tartaric acid can be combined over a range ofstoichiometries. Since tartaric acid is a diacid, molar ratios of 2:1 to1:1 IFG/tartaric acid provide stable salts (see Example 3). Thepreferred ratio is 1:1. The stoichiometry range is applicable to allisomers of tartaric acid.

The isofagomine acid salt may be purified by using most of commonly usedpurification methods, preferably recrystallization. For example, crudeisofagomine tartrate may be recrystallized in water with and withouthelp of a protic or aprotic co-solvent, preferably alcohols such as, forexample, methanol, ethanol, propanols, or butanols. Therecrystallization can be effectively conducted not only on small scalebut also on industrial scale, e.g., sub-kilogram quantities. Table 1summarizes purities and yields of several examples prepared and purifiedaccording to the present invention.

TABLE 1 Sample No. Purity (%) Yield (g) 1 >98 5 2 >98 15 3 96.8 55 484.5 45 5 95.9 40 6 87.4 71 7 94.6 45 8 95.6 343 9 95.8 851 10 99.8 1411 98.1 134 12 97.6 128 13 99.0 72 14 99.3 116 15 99.5 57 16 98.0 1368

In one preferred embodiment, crude isofagomine tartrate is dissolved inwater, and an equal amount of ethanol is added to the resulting solutionto get precipitation of the compound. Additional ethanol (1 volume) isthen added and stirred. This procedure is repeated for two additionalaliquots of ethanol to give an ethanol/water ratio of approximately 4:1.Although most of the isofagomine tartrate will crystallize after addingthe first volume of ethanol, additional aliquots can be used formaximization of the yield. After recrystallizing, the solids arefiltered and washed. The entire purification can be done at roomtemperature. Isofagomine tartrate can be purified with this method up topurity about 99% or more. Thus, isofagomine tartrate according to oneembodiment of the present invention has purity of 95% or higher,preferably 98% or higher, or even more preferably 99% or higher.

Isofagomine tartrate may also be purified using other solvents orsolvent systems such as 1:1 ethanol/water, 1:1 acetone/water, 2:1ethanol/water, 2:1 acetone/water, or 3:1 ethanol/water. Activatedcharcoal may also be used to remove any colored impurities. Each ofthese solvents can provide purities of above 95%, and most have providedpurities greater than 98%.

The ease of purification of isofagomine tartrate can be demonstrated bycomparing the purification to the purification of the HCl salt ofisofagomine in an aqueous solution, which requires lyophilization.Attempts to filter isofagomine-HCl resulted in a substance having ayellow coloration and the consistency of glue. Meanwhile, isofagominetartrate according to the present invention has good powdercharacteristics, e.g., crystal size, density, and flowability, that aresuitable for a pharmaceutical manufacturing process. Tables 2 and 3summarize powder characteristics of an isofagomine tartrate sampleprepared according to the present invention. Isofagomine tartrateprepared according to present invention is not a fine powder but ismostly populated with middle size particles with bulk density of around0.44 g/ml and Carr Index of 15%, which thus possess high flowability andeasy handling property suitable for a pharmaceutical manufacturingprocess. Isofagomine tartrate obtained according to the aboverecrystallization process also exhibits consistency in its particle sizedistribution batch to batch with a baseline span that falls between 0.7and 1.5, thus avoiding large particles, which may be detrimental toaccurate measurement of the salt during the formulation process. Mostbatches provided more than 98%, or at minimum 90%, of isofagominetartrate with a particle size of about 1200 μm or less.

TABLE 2 Sieve Analysis % Retained  40 0.6  60 15.5  80 49.2 120 28.4 2005.9 325 0.4 >325   0.0

TABLE 3 Bulk Density 0.44 g/ml Tap Density 0.52 g/ml Carr Index 15%Basline span variaton 0.79-1.53 (1.18) range for 11 batches (averagespan)

Furthermore, isofagomine tartrate is not hygroscopic, and the moistureuptake thereof was only about 0.08% after exposure to 75% RH for 8 days.The moisture uptake test results of six different isofagomine tartratesamples prepared according to present invention are summarized in Tables4 and 5.

TABLE 4 Moisture uptake studies with NaBr saturated solution. 0 hour 24hours 48 hours 8 days RH 60% 59% 59% 59% Temperature (° C.) 19.4 20.520.8 20.2 Weight Weight Weight Weight Sample No. gain (%) gain (%) gain(%) gain (%) 1 NA 0.06 0.04 0.06 2 NA 0.00 0.02 0.07 3 NA 0.05 0.10 0.10Average weight NA 0.04 0.05 0.08 gain (%)

TABLE 5 Moisture uptake studies with NaCl saturated solution. 0 hour 24hours 48 hours 8 days RH 72% 72% 71% 72% Temperature (° C.) 19.5 20.520.8 20.2 Weight Weight Weight Weight Sample No. gain (%) gain (%) gain(%) gain (%) 1 NA 0.02 0.05 0.10 2 NA 0.00 0.00 0.00 3 NA 0.08 0.8  0.15Average weight NA 0.04 0.05 0.08 gain (%)

The method for preparing isofagomine tartrate of the present inventionis thus suitable to prepare a bulk amount of isofagomine tartrate forpharmaceutical compositions. The bulk amount of isofagomine tartrateprepared according to the invention can be prepared as a slightly crudeform that having a purity of about 80% depending upon the purpose of thepreparation. However, it also could be prepared as pure as 90% or more,preferably at least 99% with the particle size of about 1200 μm or lessfor 90% of the isofagomine tartrate.

HPLC may be used to determine both the potency of isofagomine acid saltof the present invention, and the presence of organic impurities. Lowwavelength UV detection is suitable for potency calculation versus areference standard. One of skill in the art may determine properconditions for the HPLC analysis depending upon the concentration of thesample, the types of the column, solvents, etc. Nonetheless, thefollowing conditions are provided as an example for isofagominetartrate: the mobile phase may be 10 mM ammonium carbonate(NH₄HCO₃)/acetonitrile (CAN) 30/70 in isocratic mode at a flow rate of0.5 mL/min; the HPLC column may be an Alltech Prevail Carbohydratecolumn (4.6×150 mm, 5 μm particle size) operated at 50° C.; detectionmay be set at 210 nm with a 15 minute run time; and samples of drugsubstance may be dissolved in mobile phase with a 10-μL injectionvolume.

A charged aerosol detector (CAD) may be used for detection ofimpurities. Samples may also be analyzed using evaporativelight-scattering detection (ELSD) and UV detection. The CAD detectoruses evaporative technology to desolvate analytes in the presence of N₂carrier gas. A coronal spark imparts a charge to the N₂ gas, whichtransfers the charge to the analytes. Analytes are detected as theytransfer their charger to an electrometer and are measured as current.When the CAD detector is used, the mobile phase may be set to 5 mMammonium acetate/CAN 50/50 in isocratic mode at a flow rate of 1.0mL/min with a Primesep 100 (4.6×150 mm, 5 μm particle size) HPLC columnoperated at 25° C. The samples of drug substance are prepared in mobilephase, and a 10-μL injection volume may be used. The run time for thismethod is approximately 70 minutes. Impurities are determined using ahigh/low injection sequence where the sample is quantitated against thereference standard at 1% of the nominal sample concentration.

Identification of isofagomine acid salt of the present invention can beperformed using FT-IR and further confirmed by ¹H NMR and ¹³C NMR. FIGS.2-5 show ¹H, ¹³C NMR, and IR spectra of Isofagomine tartrate preparedaccording to one embodiment of the present invention. Residual solventsmay be monitored by headspace gas chromatohgraphy (GC). Water, residueon ignition, and heavy metals are monitored by standard compendialtechniques. Palladium is monitored by ICP spectroscopy as it is thecatalyst used in the final hydrogenation step.

FIG. 6 shows an X-ray powder diffraction pattern of isofagomine tartrateprepared according to one embodiment of the present invention. Thepattern was obtained using a Bruker D8 Advance diffractometer, and theanalysis was performed from 2-45° 2-theta using the followingconditions:

Divergence slit 0.6 mm Anti-scatter slit 0.6 mm Receiving slit 0.1 mmdetector slit 0.6 mm step size 0.02° step time 5 seconds

Although an X-ray powder diffractogram is useful in identifying aparticular solid form of a compound, i.e., polymorphic forms, its2-theta values as well as intensity and d-spacings may vary because ofvariations caused during the sample preparation or obtaining thepattern. Also, some margin of error is possible in the assignment of2-theta and d-spacings. The 2-theta values have a variation of ±0.009.Thus, a preferred method of comparing X-ray powder diffraction patternsin order to identify a particular crystalline form is to overlay theX-ray powder diffraction pattern of the unknown form over the X-raydiffraction pattern of a known form and to compare their characteristicpeaks. Nevertheless, the 2-theta, d-spacing, intensity and % intensityvalues of FIG. 6 are summarized in Table 6. In determining existence ofthe crystalline form of the present invention in a composition, one maycompare five or more most distinctive peaks of those identified in Table6. The most distinctive peaks include 9.29, 14.17, 16.34, 18.07, 18.72,19.44, 20.56, 22.13, 23.01, 24.54, and 27.12.

TABLE 6 Angle (2-Theta °) d value (Å) Intensity (Count) % Intensity (%) 9.29 9.5093 131 23.3 14.17 6.24684 129 22.8 16.34 5.42037 155 27.618.07 4.90414 330 58.5 18.72 4.73704 563 100 19.44 4.56252 165 29.320.56 4.31573 212 37.5 22.13 4.01417 338 60 23.01 3.86164 111 19.8 24.543.62444 210 37.2 27.57 3.23301 276 49

The Isofagomine acid salt of the present invention can be administeredin a form suitable for any route of administration, including, e.g.,orally in the form tablets, capsules, or liquid, or in sterile aqueoussolution for injection. It can be administered orally in the form oftablets, capsules, ovules, elixirs, solutions or suspensions, gels,syrups, mouth washes, or a dry powder for constitution with water orother suitable vehicle before use, optionally with flavoring andcoloring agents for immediate-, delayed-, modified-, sustained-, pulsed-or controlled-release applications. Solid compositions such as tablets,capsules, lozenges, pastilles, pills, boluses, powder, pastes, granules,bullets, or premix preparations may also be used. Solid and liquidcompositions for oral use may be prepared according to methods wellknown in the art. Such compositions may also contain one or morepharmaceutically acceptable carriers and excipients, which may be insolid or liquid form. In a specific embodiment, the isofagomine tartrateis administered as a powder-filled capsule. When the compound isformulated for oral administration, the tablets or capsules can beprepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulphate). The tablets may be coated by methodswell known in the art.

The pharmaceutically acceptable excipients also include microcrystallinecellulose, lactose, sodium citrate, calcium carbonate, dibasic calciumphosphate and glycine, disintegrants such as starch (preferably corn,potato or tapioca starch), sodium starch glycolate, croscarmellosesodium and certain complex silicates, and granulation binders such aspolyvinylpyrolidone, hydroxypropyl ethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin, and acacia. Additionally, lubricatingagents such as magnesium stearate, stearic acid, glyceryl behenate andtalc may be included.

Solid compositions of a similar type may also be employed as fillers ingelatin capsules. Preferred excipients in this regard include lactose,starch, a cellulose, milk sugar, or high molecular weight polyethyleneglycols. For aqueous suspensions and/or elixirs, the agent may becombined with various emulsifying and/or suspending agents and withdiluents such as water, ethanol, propylene glycol and glycerin, andcombinations thereof.

Liquid preparations for oral administration may take the form of, forexample, solutions, syrups or suspensions, or they may be presented as adry product for constitution with water or another suitable vehicle (forexample, ethanol or a polyol such as glycerol, propylene glycol, andpolyethylene glycol, and the like, suitable mixtures thereof, andvegetable oils) before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., water, sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol or fractionated vegetable oils); and preservatives (e.g., methylor propyl-p-hydroxybenzoates or sorbic acid). Preparations for oraladministration may be suitably formulated to give controlled orsustained release of isofagomine acid salt of the present invention.

The proper fluidity can be maintained, for example, by the use of acoating such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants. Preventionof the action of microorganisms can be brought about by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, benzyl alcohol, sorbic acid, and the like. Inmany cases, it will be reasonable to include isotonic agents, forexample, sugars or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonosterate, and gelatin.

The pharmaceutical formulations of isofagomine tartrate suitable forparenteral/injectable (for example, by intravenous bolus injection orinfusion or via intramuscular, subcutaneous or intrathecal routes) usegenerally include sterile aqueous solutions, or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. The isofagomine tartrate may be presented inunit dose forms, in ampoules, or other unit-dose containers, or inmulti-dose containers, if necessary with an added preservative. Thecompositions for injection may be in the form of suspensions, solutions,or emulsions, in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing, solubilizing, and/or dispersingagents. Alternatively, the active ingredient may be in sterile powderform for reconstitution with a suitable vehicle, e.g., sterile,pyrogen-free water, before use. In all cases, the form must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The preparation of suitable parenteral formulationsunder sterile conditions is readily accomplished by standardpharmaceutical techniques well-known to those skilled in the art.

Sterile injectable solutions are prepared by incorporating isofagominetartrate in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter or terminal sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof.

Preservatives, stabilizers, dyes, and even flavoring agents may beprovided in the pharmaceutical composition. Examples of preservativesinclude sodium benzoate, ascorbic acid, and esters of p-hydroxybenzoicacid. Antioxidants and suspending agents may be also used.

Additional pharmaceutically acceptable carriers which may be included inthe formulation are buffers such as citrate buffer, phosphate buffer,acetate buffer, and bicarbonate buffer, amino acids, urea, alcohols,ascorbic acid, phospholipids, proteins, such as serum albumin, collagen,and gelatin; salts such as EDTA or EGTA, and sodium chloride; liposomespolyvinylpyrolidone; sugars such as dextran, mannitol, sorbitol, andglycerol; propylene glycol and polyethylene glycol (e.g., PEG-4000,PEG-6000); glycerol, glycine or other amino acids and lipids. Buffersystems for use with the formulations include citrate, acetate,bicarbonate, and phosphate buffers. Phosphate buffer is a preferredembodiment.

The formulations can also contain a non-ionic detergent. Preferrednon-ionic detergents include Polysorbate 20, Polysorbate 80, TritonX-100, Triton X-114, Nonidet P-40, Octyl α-glucoside, Octyl β-glucoside,Brij 35, Pluronic, and Tween 20.

The routes for administration (delivery) include, but are not limitedto, one or more of: oral (e.g., as a tablet, capsule, or as aningestible solution), topical, mucosal (e.g., as a nasal spray oraerosol for inhalation), nasal, parenteral (e.g., by an injectableform), gastrointestinal, intraspinal, intraperitoneal, intramuscular,intravenous, intrauterine, intraocular, intradermal, intracranial,intratracheal, intravaginal, intracerebroventricular, intracerebral,subcutaneous, ophthalmic (including intravitreal or intracameral),transdermal, rectal, buccal, epidural and sublingual.

Administration of the above-described parenteral formulations ofisofagomine tartrate may be by periodic injections of a bolus of thepreparation, or may be administered by intravenous or intraperitonealadministration from a reservoir which is external (e.g., an i.v. bag) orinternal (e.g., a bioerodable implant). See, e.g., U.S. Pat. Nos.4,407,957 and 5,798,113, each incorporated herein by reference.Intrapulmonary delivery methods and apparatus are described, forexample, in U.S. Pat. Nos. 5,654,007, 5,780,014 and 5,814,607, eachincorporated herein by reference. Other useful parenteral deliverysystems include ethylene-vinyl acetate copolymer particles, osmoticpumps, implantable infusion systems, pump delivery, encapsulated celldelivery, liposomal delivery, needle-delivered injection, needle-lessinjection, nebulizer, aeorosolizer, electroporation, and transdermalpatch. Needle-less injector devices are described in U.S. Pat. Nos.5,879,327, 5,520,639, 5,846,233 and 5,704,911, the specifications ofwhich are herein incorporated by reference. Any of the formulationsdescribed above can be administered using these methods.

Furthermore, a variety of devices designed for patient convenience, suchas refillable injection pens and needle-less injection devices, may beused with the formulations of the present invention as discussed herein.

Typically, a physician will determine the actual dosage which will bemost suitable for an individual subject and will provide atherapeutically effective amount to the subject. The specific dose leveland frequency of dosage for any particular individual may be varied andwill depend upon a variety of factors including the compound activity,the type of Gaucher disease being treated, age, body weight, generalhealth, sex, diet, mode and time of administration, rate of excretion,drug combination, the severity of disease, and the individual undergoingtherapy. For oral and parenteral administration, the daily dosage levelof the agent may be in single or divided doses. Preferably, theeffective amount or dose of the isofagomine acid salt of the presentinvention is sufficient to increase the level of mutantglucocerebrosidase expression, e.g., to about 3-5%, preferably by about10%, and more preferably by about 30% of the level found in normalcells, i.e., cells from an individual not having Gaucher disease and/orcan ameliorate or prevent a clinically significant deficit GCaseactivity in the subject.

The effective amount can be often determined by routine experimentation,but is expected to be an amount resulting in serum levels between 0.01and 100 μM, preferably between 0.01 and 10 μM, most preferably between0.05 and 2 μM. The effective dose isofagomine tartrate is expected to bebetween 0.5 and 1000 mg/kg body weight per day, preferably between 0.5and 100, most preferably between 1 and 50 mg/kg body weight per day. Ina specific embodiment, the dose is between about 1-600 mg/day, morespecifically 5-300 mg/day, more specifically, 10-150 mg/day. Non-dailydosing also is contemplated. Other dosing regimens contemplated fortreatment of Gaucher disease using isofagomine tartrate are described inU.S. provisional patent application 60/914,288, filed on Apr. 24, 2007,which is herein incorporated by reference in its entirety.

The therapeutic monitoring of the present invention is also applicablefollowing treatment of patients with a combination of isofagominetartrate and another therapy such as ERT or gene therapy. Suchcombination therapy is described in commonly-owned, U.S. patentapplication publication numbers 2004/0180419 and 2004/0219132, both ofwhich are herein incorporated by reference in their entirety.

When isofagomine acid salt of the present invention is used incombination with a second therapeutic agent, the dose of each compoundmay differ from that when the compound is used alone. Appropriate doseswill be readily appreciated by those skilled in the art. It will beappreciated that the amount of a compound of the invention required foruse in treatment will vary with the nature of the condition beingtreated and the age and the condition of the patient and will beultimately at the discretion of the attendant physician.

When administration is sequential, either the compound of the inventionor the second therapeutic agent may be administered first. Whenadministration is simultaneous, the combination may be administeredeither in the same or different pharmaceutical composition.

When combined in the same formulation, it will be appreciated that thetwo compounds must be stable and compatible with each other and theother components of the formulation. When formulated separately they maybe provided in any convenient formulation, as known for such compoundsin the art.

Isofagomine may be synthesized from D-arabinose through an intermediatespreviously reported in the literature by Danishefski at al. inTetrahedron Letters. 1990; 31(16), 2229. However, the previouslyreported synthetic steps to the intermediates are not economical forlarge scale synthesis. The process disclosed herein allows for thereliable and predictable production of those intermediates (and a newintermediate) and the final product on an industrial scal and in highpurity. This is because all intermediates are isolated bycrystallization, making the process amenable to large scale production.

Isofagomine also may be synthesized the synthesis route shown in Scheme1.

D-Arabinose can be converted to the corresponding protected glycoside(A) using an appropriate alcohol with or without solvent (neatreaction), and an activating agent. For instance the range of alcoholscan include benzyl alcohol, or substituted benzyl alcohols such asmethoxybenzyl alcohol, chlorobenzyl alcohol, methanol, ethanol,isopropanol, cyclohexylmethyl alcohol and the like in a solvent such asmethylene chloride, chloroform, THF, dioxane, DMF, DMA, or NMP, with anactivating agent such as HCl, HBr, H₂SO₄, or some other mineral acid, oracetyl chloride, propionyl chloride, or another acid chloride of acarboxylic acid. The reaction can be run at temperatures ranging fromambient temperature to about 100° C., for times ranging from 2 to 48 h.For this invention the preferred alcohols are benzyl or substitutedbenzyl alcohols, and more preferred is benzyl alcohol. Preferredsolvents include dioxane, THF or neat reaction, and more preferred isneat reaction. Preferred activating agents include acetyl chloride andH₂SO₄, and more preferred is acetyl chloride. Pure product can bereadily isolated by precipitation with a non-polar solvent. Thepreferred solvent and temperature for this product is methyl-t-butylether at ambient temperature.

The obtained glycoside of general formula A can be further protected asan acetonide at the 3- and 4-hydroxyl groups by conversion of (A) toketal (B) with a ketone or a dimethylketal, or enolether thereof, in thepresence of an acid, with or without (neat) a polar co-solvent. Forinstance, aliphatic or aromatic ketones such as acetone, 2-butanone,benzophenone, cyclohexanone, or acetophenone, or their correspondingdialkylketals, can react with a vicinal diol in the presence of an acidsuch as H₂SO₄, p-toluenesulfonic acid, camphorsulfonic acid, or TMStriflate. Co-solvents include methylene chloride, DMSO, DMF, DMA, andNMP. In some cases the ketone can also be the solvent, such as acetone.Reaction temperatures can range from ambient temperature to 100° C. Forthis reaction, the preferred conditions are acetone and2,2-dimethoxypropane with p-toluenesulfonic acid at 40° C. Pure productcan readily isolated by crystallization with a two component systemincluding a polar and a non-polar component. Preferred conditions forthis purification are ethyl acetate and heptane.

The acetonide (B) can be further protected as an ether at the 2-hydroxylgroup by conversion to the corresponding alkoxide followed by subsequentreaction with an alkylating agent to provide a compound of generalformula C. Previously reported protection utilized more expensive benzylbromide and costly silver oxide. Formation of the alkoxide is readilyaccomplished with a strong base such as and alkali hydride in a polaraprotic solvent such as dialkyl ethers or THF, DMF, DMA, NMP, or DMSOcorresponding to PG2. Alkylating agents include benzyl chloride orsubstituted benzyl. Reaction temperatures can range from −20° C. to 20°C. For this reaction the preferred conditions are sodium hydride in DMFto generate the alkoxide at 0° C. to 10° C., followed by alkylation bybenzyl chloride. Pure product can be readily isolated by precipitationwith water and a non-polar wash to remove excess water. The preferrednon-polar solvent for this purification is heptane.

Removal of the acetonide in the compound of general formula C to providea diol of general formula (D) is accomplished with a dilute mineral acidsuch as HCl, HBr, H₂SO₄ in an alcohol such as methanol, ethanol,isopropanol, at ambient temperature. For this reaction, the preferredconditions are HCl in methanol at ambient temperature. Pure product (D)can be readily isolated by precipitation with water and a non-polar washto remove excess water. The preferred non-polar solvent for thispurification is heptane.

Additional protection of the diol is required for modification to thetarget molecule. Selective etherification of the 3-hydroxyl (E) can beaccomplished using a tin directed approach in a water-azeotropingsolvent at reflux temperatures followed by etherification at moderatetemperatures. Tin ethers can be formed using dialkyl or aryl tin(IV)oxides such as diphenyl, dimethyl, dibutyl, diisobutyl, or dioctyltinoxide in aprotic solvents such as benzene, toluene, or xylene.Subsequent alkylation can be accomplished with alkyl or alkylarylhalides such as benzyl bromide or benzyl chloride. The reaction can beaccelerated through the use of agents such as CsF or tetraethylammoniumchloride, and reaction temperatures can range from ambient temperatureto 100° C. For this invention the preferred method uses dibutyltin oxidein toluene and benzyl chloride in the presence of tetrabutylammoniumchloride. Purification can be readily accomplished by precipitation ofthe tin reagent with water. Final product can be obtained bycrystallization from a two solvent system. The preferred crystallizationsolvents for this reaction are ethanol and heptane.

The triprotected intermediate arabinose derivative can be directlyconverted to the corresponding xylose derivative (G) through anactivated system (F). While Mitsunobu inversion is commonly used toinvert alcohol configurations, and has been reported for this specifictransformation, those conditions are costly on a manufacturing scale. Analternative route involves activation of the arabinose hydroxyl to adiscreet, isolable activated system (G) followed by displacement withinversion using an inexpensive oxygen source. Activation can be withesters such as p-toluenesulfonate, methanesulfonate,trifluoromethanesulfonate, an the like, formed from the correspondinganhydride or sulfonyl chloride in the presence of an organic base suchas pyridine, collidine, Hunig's base, triethylamine, in a non-polarsolvent such as methylene chloride, chloroform, or toluene attemperatures from −20° C. to ambient temperature. Displacement withinversion of the configuration can be accomplished with oxygennucleophiles, preferably alkali or earth alkali metal nitrite insolvents commonly used for this type of reaction, e.g., methylenechloride, acetone, THF, DMF, DMA, NMP, and the like at temperatures from0° C. to 40° C. Preferred conditions use trifluoromethanesulfonicanhydride and pyridine in methylene chloride at −10° C. followed byisolation of the triflate without the need for purification. Preferredconditions for displacement of the triflate are sodium or potassiumnitrites in DMF at ambient temperature. Purified product can be readilyobtained by crystallization from a two solvent system using a polar anda non-polar component. The preferred crystallization solvents for thisreaction are isopropanol and heptane.

The triprotected xylose derivative of general formula (H) can beconverted into the nitrile (I) with inversion of configuration throughan activated system. Similar to the method described above, the routeinvolves activation of the xylose hydroxyl to a discreet, isolableactivated system (H) followed by displacement by a cyano source.Activation can be done again with esters of alkyl or aryl sulphonates,preferably p-toluenesulfonate, methanesulfonate,trifluoromethanesulfonate, and the like, which were formed from thecorresponding anhydride or sulfonyl chloride in the presence of a mildorganic base, such as pyridine, collidine, Hunig's base, triethylamine,and the like in a non-polar solvent such as methylene chloride,chloroform, or toluene at temperatures from −20° C. to ambienttemperature. Displacement with inversion of configuration can beaccomplished preferably with reagents such as alkali or earth alkalimetal cyanides, or tetraethylammonium cyanides in polar, aproticsolvents such as THF, DMF, DMA, NMP, DMSO, and the like at temperaturesfrom 0° C. to 40° C. Preferred conditions use trifluoromethanesulfonicanhydride and pyridine in methylene chloride at −10° C. Preferredconditions for displacement of the triflate are tetraethylammoniumcyanide in THF at ambient temperature. Purified product can be obtainedby extraction followed by crystallization from an alcoholic solvent. Thepreferred solvent is ethanol.

Conversion of the nitrile intermediate to isofagomine hydrochloride canbe carried out in one step depending on the choice of protecting groups.Nitrile reduction, triple deprotection, ring closure, and hydrogenationof the cyclic imine can be accomplished in a single step underhydrogenation conditions to provide isofagomine in high yield. Catalytichydrogenation can be carried out with a variety of common catalysts usedfor such hydrogenation including Pd/C, Pd(OH)₂/C, PtO, Degussa catalystor a combination of catalysts at loadings of 1% to 20%, under hydrogengas pressure ranging from 14 psi to 100 psi, in protic or aprotic polarsolvents, preferably alcohols such as methanol, ethanol, isopropanol, oresters, or acetic acid. The hydrogenation is carried out in the presenceof an acid such as HCl, HBr, HClO₄, H₃PO₄, H₂SO₄, acetic acid,trifluoroacetic acid, or tartaric acid. The hydrogenation can be run forshort or extended periods of time with no risk of product decomposition.Preferred conditions are to run the reaction with a mixture of Pd/C andPd(OH)₂/C with loadings of 5% to 20% under pressures from 40 psi to 100psi in an alcoholic solvent with HCl. More preferred conditions are 10%loading of Pd/C and 10% loading Pd(OH)₂/C under 80 psi hydrogen gas inethanol with HCl. This hydrochloride salt can be converted to theisofagomine acid salt of the present invention.

Another improved synthesis method for preparation isofagomine andisofagomine titrate has been recently developed and a separate patentapplication therefore has been filed. The new method utilizesD-(−)-arabinose or L-(−)-xylose through a diketal intermediate.

To illustrate further the present invention, the following examples arepresented below. The use of such examples is illustrative only and in noway limits the scope and meaning of the invention or of any exemplifiedterm. Likewise, the invention is not limited to any particular preferredembodiments described herein. Indeed, many modifications and variationsof the invention will be apparent to those skilled in the art uponreading this specification.

COMPARATIVE EXAMPLE A Purification of Isofagomine Free Base

Isofagomine free base was chromatographed using an Amberlite CG 50 resincolumn (NH₄ ⁺; 2.5 cm ID×100.0 cm L, volume 450 mL). The column waswashed with 0.5N NH₄OH solution (3 fold, 1200 mL), then with DI water (5fold, 2250 mL). Crude isofagomine (1.0 g) was dissolved in 4.0 mL waterand loaded onto the column. The column was eluted with 0.1 N NH₄OH/water(1.36:1). Fractions of 10 mL were collected.

TLC was performed on the different fractions (silica gel,isopropanol:water:NH₄OH (7:2:1) and detection was via imino sugar andninhydrin spray. Fractions testing positive with the imino-sugar sprayswere analyzed to determine purity, then combined and lyophilized for 72hours.

COMPARATIVE EXAMPLE B Purification of Isofagomine-HCl

Isofagomine free base (30 mg) was dissolved in MeOH (5 mL) and 4 N HCl(0.5 mL) in isopropanol and acetone (4.0 mL). The sample was storedrefrigerated overnight and filtered. Crystals were observed in thesolution. However, when filtered, the product was a yellow substancehaving a glue-like consistency.

This crude isofagomine-HCl was purified using an ion-exchangechromatography elating with water and ammonia.

Fractions from the ion-exchange column were concentrated bylyophilization to give a gummy semi-solid material.

EXAMPLE 1 Synthesis of IFG Tartrate From D-(−)-Arabinose

Reactions were monitored by TLC and visualized with 5% H₂SO₄/methanol,with phosphomolybdic acid solution, or with UV light at 254 nm.

Step 1: D-Arabinose (50 kg, 330.04 moles) and benzyl alcohol (132.2 kg,4.33 equivalents) were stirred and heated to 35° C. Acetyl chloride(10.9 kg, 0.42 equivalents), keeping the temperature<45° C., thenstirred 50° C. overnight. The mixture was cooled to 20° C. and dilutedwith MTBE (600 kg). The mixture was stirred for 0.5-5 h. The solids werecollected by filtration and washed with MTBE (2×40 kg). The material wasdried in a filter drier. 2-Benzyl-D-arabinose was obtained as anoff-white solid, 70.9 kg (88.6%). ¹H NMR (300 MHz, DMSO-d₆): δ 7.32 (m,5H), 4.76 (s, 1H), 4.66 (d, J=12 Hz, 1H), 4.59 (m, 3H), 4.45 (d, J=12Hz, 1H), 3.70 (m, 4H), 3.47 (dd, J=12, 3 Hz, 1H).

Step 2: 2-Benzyl-D-arabinose (73.5 kg, 305.92 moles) was mixed withacetone (522 kg). 2,2-Dimethoxypropane (26.6 kg, 1.9 equivalents) wasadded in one portion followed by p-toluenesulfonic acid monohydrate(39.3 g, 0.0007 equivalents). The mixture was stirred at 40° C. for 18hours. After the reaction was complete, triethylamine (193 mL, 0.0046equivalents) was added. The solvents were removed at 30° C. underreduced pressure until a thick oil was obtained. The residue wasco-evaporated with ethyl acetate (2×20 kg). Ethyl acetate (19.2 kg) wasadded to form a solution. Heptane (145.8 kg) was added in one portion tothe solution and cooled to −10° C. to 0° C. over night. The solids werecollected by filtration and washed with heptane (2×51.5 kg). Thematerial was dried in a filter dryer with a nitrogen purge. Theacetonide derivative(3aR,6R,7S,7aS)-6-(benzyloxy)-2,2-dimethyltetrahydro-3aH-[1,3]dioxolo[4,5-c]pyran-7-olwas obtained as an off-white solid, 70.4 kg (82%). m.p. 58-59° C. ¹H NMR(400 MHz, CDCl₃): δ 7.34 (m, 5H), 4.92 (d, J=4 Hz, 1H), 4.79 (d, J=12Hz, 1H), 4.54 (d, J=12 Hz, 1H), 4.20 (m, 2H), 4.00 (dd, J=13, 3 Hz, 1H),3.92 (dd, J=13, 2 Hz, 1H), 3.80 (m, 1H), 2.24 (d, J=7 Hz, 1H), 1.52 (s,3H), 1.35 (s, 3H).

Step 3: The acetonide derivative (78.2 kg, 278.97 moles) was mixed withDMF (295 kg, 3.77 kg/kg starting material) and cooled to 5° C. Sodiumhydride (13.4 kg, 1.2 equivalents) was added to the reactor in 3 to 4portions, maintaining the reaction mixture below 10° C. then stirred for1.5 hours. At a temperature of 2° C., benzyl chloride (45.9 kg, 1.3equivalents) was added over a 1 hour period. The reaction was stirred at10° C. to 15° C. for 12 h. After the reaction was complete, the mixturewas cooled to 2° C. and water (20 kg) was added over 1 h. An additionalcharge of water (570 kg) was added over 4 hours. The mixture was stirredat this temperature for 10 h. The product was collected by centrifugefiltration and washed with water (2×10 kg) and heptane (2×15 kg) spundry overnight. The dibenzyl derivative(3aR,6R,7S,7aR)-6,7-bis(benzyloxy)-2,2-dimethyltetrahydro-3aH-[1,3]dioxolo[4,5-c]pyranwas obtained as a white solid, 74.0 kg (71.6%).

Step 4: The dibenzyl derivative (37.6 kg, 101.50 moles) was added tomethanol, AR (259 kg, 8.7 kg/kg starting material) and the contents werecooled to 15° C. A 2.5 N HCl solution (76.2 kg, 1.8 equivalents) wasadded over 1 hour. Additional water (20 kg) was added and the mixturewas stirred for 12 hours at 15° C. Water (1035 kg, 4× vol methanol, AR)was added to the reactor and stirred for at least 0.5 h. The product wasfiltered onto a centrifuge and washed with water (2×10 kg) and heptane(2×15 kg) and spun dry overnight. The diol(3R,4R,5S,6R)-5,6-bis(benzyloxy)tetrahydro-2H-pyran-3,4-diol wasobtained as a white solid, 31.5 kg (94%).

Step 5: The diol derivative (37.5 kg, 113.51 moles) was mixed withtoluene (207.6 kg, 5.5 kg/kg of diol) and dibutyltinoxide (31.1 kg, 1.1equivalents). The reactor was equipped with a Dean-Stark apparatus andthe reactor contents were heated to reflux (approx. 110° C.) until waterno longer collected for removal (8-12 h). The reactor contents werecooled to 35° C. and tetrabutylammonium chloride (18.3 kg, 0.5equivalents) was added in one portion. Benzyl chloride (15.8 kg, 1.1equivalents) was added at a rate that kept the temperature<40° C. andstirring continued at 35° C. for 12 h. The addition and 12 h stirringwere repeated daily for 4 days until the reaction was complete. Afterthe reaction was complete, the mixture was cooled to 25° C., water (150kg) was added in one portion, and the contents were stirred overnight.The reaction mixture was filtered through a bed of Celite (1 kg/kg ofdiol) and the bed was rinsed with toluene (10 kg). The filtrate wasallowed to settle (1 h) and the layers were separated. Water addition,stirring, filtration, and separation were repeated. The aqueous layerswere combined and extracted with ethyl acetate (25 kg), and the layerswere separated. The organic layers were combined and concentrated undervacuum at 45° C. to a minimum stirable volume. Heptane (102.6 kg) wasadded. The mixture was stirred for 20 minutes, cooled to 0° C., andstirred for 8-12 h. The solids were collected by filtration and washedwith heptane (10 kg). Crude solids were dissolved in 6:1 heptane/200 pfethanol (7 kg/kg crude solid) at 35° C., cooled to −5° C. to 0° C. andstirred overnight. The solids were collected by filtration and washedwith heptane (10 kg). The product purity was monitored by TLC.Typically, 2 or more re-crystallizations were required to remove theimpurities. The purified tribenzyl derivative was dried in a vacuum ovenat 30° C. (3R,4R,5S,6R)-4,5,6-Tris(benzyloxy)tetrahydro-2H-pyran-3-olwas obtained as a white solid, 17.5 kg (37%). m.p. 59-60° C. ¹H NMR (400MHz, CDCl₃): δ 7.38 (m, 15H), 4.89 (d, J=4 Hz, 1H), 4.82 (d, J=12 Hz,1H), 4.71 (m, 3H), 4.57 (d, J=12 Hz, 1H), 4.55 (d, J=12 Hz, 1H), 4.01(br s, 1H), 3.95 (dd, J=10, 3 Hz, 1H), 3.83 (m, 2H), 3.71 (dd, J=12, 2Hz, 1H), 2.56 (brs, 1H).

Step 6: The tribenzylarabinose derivative (12.0 kg, 28.54 moles) wasmixed with methylene chloride (79.2 kg, 6.6 kg/kg starting material) andpyridine (11.3 kg, 5 equivalents) and cooled to −10° C.Trifluoromethanesulfonic anhydride (10.1 kg, 1.25 equivalents) was addedat a rate that kept the temperature below 0° C. The reaction mixture wasstirred at −10° C. to 0° C. until starting material was consumed. Oncecomplete, the reaction mixture was washed with 7.5% HCl solution (3×68kg, 17 equivalents) and water (48 kg). During the washes, thetemperature of the reaction mixture was maintained at <5° C. The mixturewas adjusted to pH≧6 by washing with 7.5% NaHCO₃ solution (55.0 kg).Triethylamine (0.4 kg, 0.3 kg/kg starting material) was added and theorganic phase was dried with anhydrous K₂CO₃ (1.2 kg, 0.1 equivalents).The mixture was filtered and concentrated to dryness under vacuum at 20°C. to 35° C. to give(3S,4R,5S,6R)-4,5,6-Tris(benzyloxy)tetrahydro-2H-pyran-3-ol. Thetriflate was used without purification. ¹H NMR (300 MHz, CDCl₃): δ7.31-7.16 (m, 15H), 5.12 (br s, 1H), 4.83 (d, J=4 Hz, 1H), 4.76 (d, J=11Hz, 1H), 4.64 (m, 2H), 4.50 (d, J=9 Hz, 1H), 4.46 (d, J=8 Hz, 1H), 3.97(dd, J=10, 3 Hz, 1H), 3.86 (d, J=14 Hz, 1H), 3.77-3.72 (m, 2H).

Step 7: The triflate was dissolved in DMF (36.2 kg, 3.02 kg/kg startingmaterial) and cooled to 10° C. Sodium nitrite (5.9 kg, 3.0 equivalents)was added, the solution exothermed to approximately 30° C., then thereaction mixture was cooled to 15° C. to 25° C. and stirred for 12-16 h.The mixture was cooled to 5° C., and water (152 kg, 4.2 kg/kg DMF) wasadded at a rate that kept the temperature<15° C. The resulting mixturewas agitated at 10° C. for 2 hours. The solids were filtered and washedwith water (2×12 kg). The filtered solids were dissolved in ethylacetate (21.6 kg, 1.8 kg/kg starting material). The solution was washedwith brine (15.5 kg), dried with MgSO₄ (2.5 kg), filtered, and thefiltrate was concentrated to dryness under vacuum at 35° C. Isopropanol(9.5 kg) was added and heated to 75° C. to dissolve the crude product.Heptane (24.6 kg) was added to the solution and the mixture cooled to15° C. to 25° C. The mixture was further cooled to 0° C. and stirredovernight. The solids were filtered and washed with heptane (2×8.2 kg).The material was dried in a vacuum oven.(3S,4R,5S,6R)-4,5,6-Tris(benzyloxy)tetrahydro-2H-pyran-3-ol was obtainedas a yellow solid, 5.3 kg (44%). ¹H NMR (300 MHz, CDCl₃): δ 7.37 (m,15H), 4.96 (d, J=11 Hz, 1H), 4.80 (m, 2H), 4.68 (d, J=12 Hz, 1H), 4.61(m, 2H), 4.53 (d, J=12 Hz, 1H), 3.78 (m, 1H), 3.67 (m, 3H), 3.50 (dd,J=9, 3 Hz, 1H), 2.42 (br s, 1H).

Step 8: The tribenzylxylose derivative (10.4 kg, 24.73 moles) was mixedwith methylene chloride (68.6 kg, 6.6 kg/kg starting material) andpyridine (9.8 kg, 5 equivalents) and cooled to −10° C.Trifluoromethanesulfonic anhydride (8.7 kg, 1.25 equivalents) was addedat a rate that kept the temperature below 0° C. The reaction mixture wasstirred at −10° C. to 0° C. until starting material was consumed. Oncecomplete, the reaction mixture was washed with 7.5% HCl solution (3×58.9kg, 17 equivalents) and water (41.6 kg). During the washes, thetemperature of the reaction mixture was maintained at <5° C. The mixturewas adjusted to pH>6 by washing with saturated NaHCO₃ solution (44.6kg). Triethylamine (0.4 kg, 0.3 kg/kg starting material) was added andthe organic phase was dried with anhydrous K₂CO₃ (1.2 kg, 0.1equivalents). The mixture was filtered and concentrated to dryness undervacuum at 20° C. to 35° C. NMR?

Step 9: The triflate was dissolved in THF (29 kg, 2.8 kg/kg startingmaterial) and cooled to 10° C. Tetraethylammonium cyanide (4.6 kg, 1.2equivalents) was added, the solution exothermed, then the reactionmixture was cooled to 20° C. and stirred for 12 h. Ethyl acetate (21.8kg) was added and the organic phase was washed with 10% NaCl solution(3×14.3 kg). The combined aqueous layers were extracted with ethylacetate (21.8 kg). The organic layers were combined, dried with MgSO₄ (2kg), filtered, and concentrated to dryness under. Ethanol (200 pf, 3.23kg/kg starting material) was added and heated to 70° C. to dissolve thecrude product. The solution was cooled to 20° C., then further cooled to5° C. and stirred overnight. The solids were filtered and washed withheptane (2×10.4 kg). Crystallization from 200 pf ethanol (7 mL/g solids)was repeated. The solids were filtered and washed with heptane (2×10.4kg). The material was dried in a vacuum oven.(3R,4R,5S,6S)-4,5,6-Tris(benzyloxy)tetrahydro-2H-pyran-3-carbonitrilewas obtained as a light brown solid, 6.3 kg (59%). ¹H NMR (300 MHz,CDCl₃): δ 7.31 (m, 15H), 4.90 (d, J=3 Hz, 1H), 4.81-4.73 (complex, 3H),4.70 (d, J=12 Hz, 1H), 4.62 (d, J=12 Hz, 1H), 4.55 (d, J=12 Hz, 1H),3.99 (dd, J=9, 5 Hz, 1H), 3.91 (dd, J=12, 3 Hz, 1H), 3.82-3.74(overlapping signals, 2H), 3.13 m, 1H).

Step 10: The nitrile derivative (2.5 kg, 5.8 moles) was dissolved inabsolute ethanol (138.1 kg) and heated at 35° C. until a clear solutionwas obtained. Moistened palladium on carbon was added (250 g; 10% w/w),followed by palladium hydroxide, (250 g; 20% w/w) and hydrochloric acid(0.6 L). The solution was purged twice with nitrogen and once withhydrogen. The solution was pressurized to 80 psi with hydrogen, stirred,and heated to 35° C. for 72 hours, repressurizing as necessary. Themixture was filtered and concentrated under vacuum at 30° C. to 35° C.Crude isofagomine hydrochloride was mixed with aq NH₄OH (3 L). Thesolution was filtered and purified on a silica gel column (approx 20 kg)using 9:1 EtOH/aq NH₄OH solvent system. The product was concentratedunder vacuum at 35° C. to 40° C. The isofagomine free base was dissolvedin absolute ethanol (7.7 mL/g residue) and filtered. L-(+)-Tartaric acid(1185 g, 1 g/g residue) was dissolved in absolute ethanol (7.7 mL/gresidue), filtered, and slowly added to the solution of isofagomine inethanol. This solution was stirred for 45 minutes, filtered, and washedwith ethanol (2.5 L, 1 mL/g starting material). The product was dried toconstant weight in a vacuum oven at 44° C. Isofagomine tartrate wasobtained as a white solid 1.2 kg (97.5% purity). ¹H NMR (300 MHz, D₂O):δ 4.40 (s, 2H), 3.70 (dd, J=12, 4 Hz, 1H), 3.66-3.58 (m, 2H), 3.38 (m,3H), 2.83 (t, J=13 Hz, 1H), 2.79 (t, J=13 Hz, 1H), 1.88-1.77 (m, 1H).

EXAMPLE 2 Recrystallization of Isofagomine Tartrate

Isofagomine tartrate (1,767 g) was dissolved in water (1.767 L) atambient temperature. Absolute EtOH (1.767 L) was added and stirred forover 30 minutes. An additional aliquot of absolute EtOH (1.767 L) wasadded dropwise at a slow stream and stirred for 30 minutes. This processwas repeated twice (including the 30-minute of stirring) to obtain asolution of 4:1 EtOH/water. The solids were filtered and washed withEtOH/water (4:1) and dried in a vacuum oven at 43° C. overnight to aconstant weight (i.e., <1% net weight loss after re-drying for anadditional 2 hours). The yield from recrystallization was 91%. Thesample was found to have 1.3% impurities based on HPLC. NMR spectra ofthe recrystallized product are shown in FIG. 3 and FIG. 4. m.p. 168-169°C.

EXAMPLE 3 Synthesis of Isofagomine Tartrate Salts

Isofagomine L(+)-Tartrate Salt (2:1)

A solution of L-(+)-tartaric acid (102 mg, 0.679 mmol) in deionizedwater (1.0 mL) was added into the solution of isofagomine (200 mg, 2.0equivalents) dissolved in deionized water (2.0 mL) at room temperature.The solution was stirred for 10 min and lyophilized overnight. Theresidue was further dried under vacuum at 45° C. for three days toafford the desired salt (275.6 mg, 91%). m.p. 92-93° C., ¹H NMR (300MHz, D₂O): δ 4.22 (s, 2H), 3.71 (dd, J=12, 3.6 Hz, 1H), 3.67-3.59 (m,2H), 3.44-3.37 (m, 3H), 2.85 (t, J=12 Hz, 1H), 2.75 (t, J=12 Hz, 1H),1.85 (m, 1H) (FIG. 8A)

Isofagomine D-(−)-Tartrate Salt (2:1)

A solution of D-(−)-tartaric acid (102 mg, 0.679 mmol) in deionizedwater (1.0 mL) was added into the solution of isofagomine (200 mg, 2.0equivalents) dissolved in deionized water (2.0 mL) at room temperature.The solution was stirred for 10 min and lyophilized overnight. Theresidue was further dried under vacuum at 45° C. for three days toafford the desired salt (287.2 mg, 95%). m.p. 94-95° C., ¹H NMR (300MHz, D₂O): δ 4.22 (s, 2H), 3.71 (dd, J=12, 3.6 Hz, 1H), 3.67-3.59 (m,2H), 3.44-3.36 (m, 3H), 2.85 (t, J=12 Hz, 1H), 2.75 (t, J=12 Hz, 1H),1.84 (m, 1H) (FIG. 8B).

Isofagomine D-(−)-Tartrate Salt (1:1)

A solution of D-(−)-tartaric acid (204 mg, 1.359 mmol) in deionizedwater (2.0 mL) was added into the solution of isofagomine (200 mg, 2.0equivalents) dissolved in deionized water (2.0 mL) at room temperature.The solution was stirred for 10 min and lyophilized overnight. Theresidue was further dried under vacuum at 45° C. for three days toafford the desired salt (396.9 mg, 98%). m.p. 73-74° C., ¹H NMR (300MHz, D₂O): δ 4.41 (s, 2H), 3.71 (dd, J=12, 3.3 Hz, 1H), 3.66-3.59 (m,2H), 3.44-3.36 (m, 3H), 2.84 (t, J=12 Hz, 1H), 2.75 (t, J=12 Hz, 1 H),1.84 (m, 1H) (FIG. 8C)

EXAMPLE 4 Formulations of Isofagomine Tartrate Capsule

10 mg Capsule, Prototype 1 100 mg Capsule, Prototype 2 % w/w mg/capsuleg/batch % w/w mg/capsule g/batch Isofagomine 5.50 10.00 15.00 50.00100.00 40.00 tartrate Avicel PH102 ® 94.00 170.79 256.19 49.50 99.0039.60 (microcrystalline cellulose) Magnesium .050 .091 1.36 0.50 1.00.040 Stearate Total 100.00 181.70 272.55 100.00 200.00 80.00 opaquewhite 1500 capsules 400 capsules capsule shells, (estimated shell size =2 or 3) (estimated shell size = 2 or 3) size tbd

10 mg Capsule, Prototype 3 100 mg Capsule, Prototype 4 % w/w mg/capsuleg/batch % w/w mg/capsule g/batch Isofagomine 4.35 10.00 15.00 40.00100.00 40.00 tartrate Emcompress ® 47.43 109.08 163.62 29.60 74.00 29.60(dibasic calcium phosphate) Avicel PH102 ® 47.43 109.08 163.62 29.6074.00 29.60 (microcrystalline cellulose) Cab-O-Sil ® .30 .69 1.04 .30.75 .30 (colloidal (fumed) silicon dioxide) Magnesium 0.50 1.15 1.73.050 1.25 .050 Stearate Total 100.00 230.00 345.00 100.00 250.00 100.00opaque white 1500 capsules 400 capsules capsule shells, (estimated shellsize = 2 or 3) (estimated shell size = 2 or 3) size tbd

EXAMPLE 5 Intracellular Enhancement of GCase Activity in Fibroblastsfrom Gaucher Patients

The intracellular enhancement activity of isofagomine L-(+)-tartrate wasinvestigated with fibroblasts established from Gaucher patients. Toevaluate the effects of IFG on mutant GCase levels, an ex vivo responsestudy with macrophages and EBV-transformed lymphoblasts derived fromperipheral leukocytes of 40 patients was conducted.

The study included 21 males with type I Gaucher disease, 1 male withtype III Gaucher disease, and 18 females with type I Gaucher disease.Patients ranged in age from 7 to 83 years, and 38 of 40 patients were onenzyme replacement therapy (ERT). Macrophages were successfully derivedfrom 34 of 40 patients, of which 32 demonstrated a dose-dependentincrease in GCase levels (average=2.8-fold) when treated with IFGtartrate (5 days). Similar results were observed for 5 additionalpatient-derived lymphoblast cell lines. IFG significantly increasedGCase levels in cells from patients with different genotypes includingN370S/N370S (11), N370S/L444P (8), N370S/84insG (11), N370S/R163X,N370S/Y212H, L444P/del 136T, L444P/F216Y, L444P/L174F, G202R/R463C, andK79N/complex B exon 9/10 (type III GD). Maximum enhancement of GCase inmacrophages was achieved at about 30 μM of IFG.

EXAMPLE 6 Phase I Studies of the Safety, Pharmacokinetics andPharmacodynamics of Isofagomine Tartrate, a New PharmacologicalChaperone for the Treatment of Gaucher Disease

Isofagomine tartrate is a pharmacological chaperone in development forthe treatment of the lysosomal storage disorder Gaucher disease. Usingcell-based and animal models we have shown that isofagomine increasescellular levels of glucocerebrosidase (GCase), the enzyme deficient inGaucher disease. Randomized double-blind Phase I clinical studies wereperformed in 72 healthy volunteers, (39 male, 33 female). Isofagominetartrate was orally administered as an aqueous solution. In afirst-in-human single ascending dose study, doses of 8, 25, 75, 150 (twocohorts), and 300 mg were administered (6 active, 2 placebo in eachcohort). In a multiple ascending dose study, doses of 25, 75, and 225 mgwere administered daily for seven days (6 active, 2 placebo in eachcohort). In both studies, isofagomine tartrate was generally welltolerated at all doses and treatment-emergent adverse events in bothstudies were mostly mild. No serious adverse events occurred.

Isofagomine tartrate showed good systemic exposure via the oral route.In the single-dose study, plasma AUC and C_(max) values were linearlycorrelated with administered dose. Mean plasma levels peaked at 3.4 hr.(SEM: 0.6 hr.) and the plasma elimination half-life was 14 hr. (SEM: 2hr.). In the multiple-dose study, after 7 days of oral administration,the pharmacokinetic behavior was found to be linear with dose, with nounexpected accumulation of isofagomine. T_(max) and half-life valueswere similar to those observed in the single-dose study.

In the multiple-dose study, GCase activity in isolated white blood cellswas measured at days 1, 3, 5 and 7 during administration of isofagominetartrate, and at days 9, 14 and 21 during the post-treatment washoutperiod. In all subjects receiving isofagomine tartrate there was amarked increase in GCase levels during the treatment period, followed bya decrease upon removal of the drug and a return to near baseline levelsby day 21 (FIG. 9). The increase in enzyme level was dose-related,reaching approximately 3.5-fold above baseline levels. These results forthe safety, pharmacokinetics and preliminary pharmacodynamic effects inhealthy volunteers support the further evaluation of isofagominetartrate for the treatment of Gaucher disease.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that all values are approximate, and areprovided for description.

Patents, patent applications, publications, product descriptions, andprotocols are cited throughout this application, the disclosures ofwhich are incorporated herein by reference in their entireties for allpurposes.

What is claimed is:
 1. A method for the preparation of isofagominetartrate comprising: (a) reacting isofagomine free base or its ionizedform and L-(+)-tartaric acid in a molar ratio of 1:1 in a solvent; and(b) isolating isofagomine tartrate.
 2. The method of claim 1, wherein atleast one kilogram of isofagomine tartrate is isolated.
 3. The method ofclaim 1, wherein the solvent is an aqueous alcohol.
 4. The method ofclaim 1, wherein the solvent is an alcohol selected from methanol,ethanol, propanols, butanols, or mixtures thereof.
 5. The method ofclaim 1, wherein the solvent is ethanol.
 6. The method of claim 1,wherein step (b) includes crystallization of isofagomine tartrate. 7.The method of claim 6, wherein 90% or more of isofagomine tartrate has aparticle size of 1200 μm or less following crystallization ofisofagomine tartrate.
 8. The method of claim 6, wherein crystallizationof isofagomine tartrate comprises water and a secondary solvent.
 9. Themethod of claim 8, wherein the secondary solvent is a protic solvent.10. The method of claim 9, wherein the protic solvent is an alcohol. 11.A method for preparing high purity isofagomine tartrate on an industrialscale, the method comprising, (a) reacting about 1 kg or moreisofagomine free base or its ionized form and L-(+)-tartaric acid in amolar ratio of 1:1 in a solvent; (b) obtaining a crude isofagominetartrate; (c) crystallizing the crude isofagomine tartrate using anaqueous solution; and (d) isolating high purity isofagomine tartrate.12. The method of claim 11 wherein the aqueous solution is water. 13.The method of claim 11, wherein the aqueous solution is a mixture ofwater and an alcohol wherein water and the alcohol are used at the sametime or with an interval of time.
 14. The method of claim 11, whereinhigh purity isofagomine tartrate is at least 84% pure by weight.
 15. Themethod of claim 11, wherein high purity isofagomine tartrate is at least98% pure by weight.
 16. The method of claim 11, wherein high purityisofagomine tartrate is at least 99% pure by weight.
 17. The method ofclaim 11, wherein 90% or more of high purity isofagomine tartrate has aparticle size of 1200 μm or less.
 18. A method for the preparation ofisofagomine tartrate comprising: (a) reacting isofagomine free base orits ionized form and L-(+)-tartaric acid in a molar ratio of 2:1 to 1:1in a solvent; and (b) isolating isofagomine tartrate.
 19. The method ofclaim 18, wherein at least one kilogram of isofagomine tartrate isisolated.
 20. The method of claim 18, wherein the solvent is an aqueousalcohol.
 21. The method of claim 18, wherein the solvent is an alcoholselected from methanol, ethanol, propanols, butanols, or mixturesthereof.
 22. The method of claim 18, wherein the solvent is ethanol. 23.The method of claim 18, wherein step (b) includes crystallization ofisofagomine tartrate.
 24. The method of claim 23, wherein 90% or more ofisofagomine tartrate has a particle size of 1200 μm or less followingcrystallization of isofagomine tartrate.
 25. The method of claim 23,wherein crystallization of isofagomine tartrate comprises water and asecondary solvent.
 26. The method of claim 25, wherein the secondarysolvent is a protic solvent.
 27. The method of claim 26, wherein theprotic solvent is an alcohol.
 28. A method for preparing high purityisofagomine tartrate on an industrial scale, the method comprising, (a)reacting about 1 kg or more isofagomine free base or its ionized formand L-(+)-tartaric acid in a molar ratio of 2:1 to 1:1 in a solvent; (b)obtaining a crude isofagomine tartrate; (c) crystallizing the crudeisofagomine tartrate using an aqueous solution; and (d) isolating highpurity isofagomine tartrate.
 29. The method of claim 28 wherein theaqueous solution is water.
 30. The method of claim 28, wherein theaqueous solution is a mixture of water and an alcohol wherein water andthe alcohol are used at the same time or with an interval of time. 31.The method of claim 28, wherein high purity isofagomine tartrate is atleast 84% pure by weight.
 32. The method of claim 28, wherein highpurity isofagomine tartrate is at least 98% pure by weight.
 33. Themethod of claim 28, wherein high purity isofagomine tartrate is at least99% pure by weight.
 34. The method of claim 28, wherein 90% or more ofhigh purity isofagomine tartrate has a particle size of 1200 μm or less.